The immune system is central to control of infectious diseases and cancer. Lymphocytes, a class of white blood cells, are critical cell types that are responsible for the activities of the immune system. Lymphocytes are divided into three major categories, T lymphocytes, NK cells and B lymphocytes. Overall assessment of the function of lymphocytes is important in determining the status of the immune system, in particular, immunodeficiency caused by genetic factors, infectious disease such as (HIV), drugs following transplantation, stress, aging, or nutritional deprivation.
Lymphocytes, which are usually in a metabolically inactive or resting state in the peripheral blood, express receptors on the cell surface that bind specific antigens. Exposure to an antigen results in metabolic activation and subsequent expansion of the population of the lymphocytes that are reactive to that antigen (clonal or oligoclonal response). Measurement of the response of the immune system to a specific antigen can be useful, for example, in diagnosis of infectious disease, hypersensitivity to certain agents, exposure to immunologically reactive drugs, assessing responses to vaccination or responses to an organ or tissue transplant. However, it is difficult to measure clonal responses since very few clones of cells (about 1 in 106 may exist in a ml of blood) may respond to an antigen. This has led to assays where a polyclonal activator, such as Phytohemagglutinin (PHA), is used as a surrogate stimulant thus allowing the detection of increased function in a larger number of cells.
The function of B lymphocytes or their response to a specific antigen can be assessed by measuring the level of specific antibody in bodily fluids such as blood, saliva or urine. However methods of detecting antibody from a single clone of B lymphocytes in peripheral blood are difficult and time consuming. The function of T lymphocytes or their response to specific antigens is also difficult to measure. Measurement of the functions of T lymphocytes or T cells is complicated by a number of factors. First, there are several different subsets of T cells with different functions. These subsets have been classified in part by the expression of characteristic cell surface markers and in part by a variety of functional assays including measurement of cytokines. Second, T cells respond to antigens only when they are presented by other cells in the context of major histocompatibility antigens on the surface of the presenting cell. Third, many of the functions of T cells depend on cell-cell contact with effector cells or the functions are fairly localized. Current methods for measuring immune function are tedious, time consuming, and poorly adapted to the clinical laboratory setting.
Methods that are currently used for measurement of immune function include: methods based on measuring the increase in activation markers, methods based on measuring the proliferation of lymphocytes, methods based on measuring DNA synthesis, methods based on measurement of cytotoxic activity or secretion of cytokines or other soluble factors, and methods used in vivo, such as skin tests.
Direct measurement of responses of lymphocytes has included proliferation assays, cytotoxicity assays, and measurement of cytokines. In general, these methods require separation of white cells from the original sample followed by incubation with antigen or mitogen. Measurement of the function of specific subsets of lymphocytes requires extensive and expensive manipulations prior to the assay. The requirement for antigen presenting cells then means that additional cells have to be added back to the culture. Lympho-proliferation assays are based on division of responding cells and are typically performed by incorporation of radioactive or nonradioactive labeled precursors of DNA synthesis. DNA synthesis or other proliferation assays take 2-10 days and are subject to significant variability based on the specific technique and the reagents used in the assay. Cytotoxic tests also require significant cell manipulation and time, and require appropriate target cells.